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miRNA-29 bioseparation and target delivery strategies for Alzheimer's disease

Publication . Pereira, Patrícia Alexandra Nunes; Sousa, Fani Pereira de; Figueiras, Ana Rita; Correia, Ilídio Joaquim Sobreira

The possibility of selectively alter the expression pattern of a particular gene has been sought by scientists and clinicians for a long time. Nowadays, RNA interference (RNAi)-based technology has become a novel tool for silencing gene expression in cells. In addition, this strategy encloses an enormous therapeutic potential that could change the course of the currently applied treatments in several life threatening pathologies and it is expected that this technology can be translated onto clinical applications in a near future. MicroRNA (miRNA) has become a commonly employed tool for gene silencing, since it prevents protein synthesis by inducing the messenger RNA (mRNA) degradation, with a high specificity degree. Consequently, in the last years, the miRNAs have emerged as biopharmaceuticals to regulate several pathways involved in the insurgence and progression of the Alzheimer’s disease (AD), since they might have key regulatory roles in many neuronal functions, such as differentiation, synaptic plasticity and memory formation, and typically they are down-regulated in disease conditions. In the literature there are some studies describing a causal relationship between miR-29 expression and AD, since a loss of miR-29 cluster can contribute to increased beta-amyloid precursor protein-converting enzyme 1 (BACE1) and Amyloid-β (Aβ) levels in sporadic AD patients. Thus, this evidence supports the possibility to use miR-29 as a potential therapeutic target for AD therapy. In general, miRNA-based therapy relies on the use of synthetic microRNAs. However, these synthesized formulations typically present contaminants that can lead to non-targeted gene silencing, which still restricts the pre-clinical or clinical application of these RNAs. Thus, considering this therapeutic purpose and the global distribution of novel biopharmaceuticals it is necessary to develop efficient processes for their preparation. The development of new strategies for microRNA production with high purity degree and biologically active is extremely required. One of the strategies might be the use of the recombinant production of biomolecules using prokaryotic hosts. Hence, the present work intends to develop and establish an integrative biotechnological platform to biosynthesize and purify a recombinant miRNA precursor (pre-miR-29b) to act in the selective silencing of endogenous pathways directly related with AD, in particular BACE1 and Aβ. In addition, the success of these therapies also depends upon the ability to selectively and efficiently deliver the pre-miR-29b in the cytoplasmic compartment of neuronal cells, the location where their function is exerted; therefore the development of miRNA delivery systems was also envisioned. The expression system Rhodovulum sulfidophilum (R. sulfidophilum) DSM 1374 allowed, for the first time, the production of human pre-miR-29b with a straightforward recuperation of pre-miR-29b in a single step, maintaining its biological active form. The application of this recombinant bacterial microorganism is innovative and is supported by the unusual capacity of secreting the nucleic acids to the extracellular space and the absence of host ribonucleases in the culture medium. Therefore, it is expected that the secreted miRNA will be devoid of main bacterial associated impurities. Regarding the growth conditions, and conversely to what was previously described for this bacterium, our results showed to be possible to develop an original approach for the aerobic growth of the R. sulfidophilum, which results in a cell growth improvement followed by an enhanced production of human pre-miR-29b. The extracellular pre-miR-29b concentration was approximately 182 μg/L, after 40 hours of bacterial growth and the total intracellular pre-miR-29b was of about 358 μg/L, at 32 hours of cell growth. To further develop a potential therapeutic application, the major interest is not only to produce high quantities of RNA but also to obtain and preserve its biological active form, fulfilling the requirements of regulatory agencies. Hence, to assure that this prerequisite is met it was used a novel and effective purification strategy, based on affinity chromatography, to purify the pre-miR-29b. Therefore, in order to achieve the selectivity towards the target pre-miRNA and the maximum resolution between the pre-miR-29b and other host biomolecules (transfer RNAs and proteins) it was used an affinity support that exploits the same biological interactions that are established within the cell, by using immobilized amino acids (L-lysine and L-arginine), as specific ligands. The recognition of the pre-miR-29b achieved with these supports, allowed its selective recovery from a complex mixture with high efficiency and high purity. In parallel, the binding of pre-miRNA to these different amino acids was studied by Surface Plasmon Resonance. This information brings important insights concerning the characterization of the pre-miRNA binding onto chromatographic supports. Moreover, it was possible to determine some particular conditions enabling the improvement of the binding specificity of the amino acid ligands used to purify miRNA, preserving the RNA integrity. Taking into account that the structure of the chromatographic supports has been continuously developed to afford rapid and efficient separations, namely for the purification of nucleic acids, it was also tested a monolithic support to purify the pre-miR-29b. The association of the high capacity of these supports with the specificity conferred by the agmatine ligand (a derivative of L-arginine) represented a novelty and an advantage to obtain highly pure pre-miR-29b (90%) with a high recovery yield (95%). The establishment of an effective application of miRNAs is usually constrained by different phenomena, namely their easy degradation when in contact with the body fluids. To overcome this limitation, delivery systems, such as polymeric systems (polyplexes), were developed and characterized in order to encapsulate and protect the pre-miR-29b biopharmaceuticals from degradation, allowing their sustained and targeted release. The formulations prepared with chitosan and polyethylenimine demonstrated high loading capacity, small sizes and exhibited a strong positive charge on their surface. In addition, considering the application field of this work, the delivery systems should also have the ability to penetrate the Blood-Brain Barrier (BBB), causing an increase of the pre-miRNAs concentration in the brain and, consequently the improvement of the therapeutic effect. Actually, BBB is an intrinsic barrier limiting miRNA therapeutic effect on the central nervous system. Thus, to improve the delivery of pre-miRNA therapeutics in the brain, the polyplexes were functionalized with specific ligands, namely lactoferrin and stearic acid which are recognized by cell surface receptors of BBB. Finally, it was evaluated the biological activity of the recombinant pre-miR-29b by measuring the efficiency on human BACE1 knockdown, using in vitro neuronal cell lines. The effect of recombinant pre-miR-29b administration was verified by both assessing the mRNA and protein human BACE1 levels, by using RT-qPCR, Western blot and Imunocytochemistry. Results suggest that recombinant pre-miR-29b can represent a novel biopharmaceutical product for the therapeutic modulation of human BACE1 levels, because high levels of inhibition were achieved, namely 80% of reduction for BACE1 protein expression and 45% for Aβ42 levels. Globally, the implementation of these cutting-edge technologies can have a great impact on the biopharmaceutical industry, providing the basis for the implementation of novel miRNA-based therapeutics, not only for neurological disorders but also for future therapeutic targets that can be of potential interest.

2016Doctoral thesis

Open access

Chromatographic HPV-16 E6/E7 plasmid vaccine purification employing L-histidine and 1-benzyl-L-histidine affinity ligands

Publication . Amorim, Lúcia; Gaspar, Rita; Pereira, Patrícia; Černigoj, Urh; Sousa, Fani; Queiroz, João; Sousa, Ângela

Affinity chromatography based on amino acids as interacting ligands was already indicated as an alternative compared to ion exchange or hydrophobic interaction for plasmid DNA purification. Understanding the recognition mechanisms occurring between histidine-based ligands and nucleic acids enables more efficient purification of a DNA vaccine, as the binding and elution conditions can be adjusted in order to enhance the purification performance. Decreasing pH to slightly acidic conditions increases the positive charge of histidine ligand, what influences the type of interaction between chromatographic support and analytes. This was proven in this work, where hydrophobic effects established in the presence of ammonium sulfate were affected at pH 5.0 in comparison to pH 8.0, while electrostatic and cation-π interactions were intensified. Histidine ligand at pH 5.0 interacts with phosphate groups or aromatic rings of plasmid DNA. Due to different responses of RNA and pDNA on mobile phase changes, the elution order between RNA and pDNA was changed with mobile phase pH decrease from 8.0 to 5.0. The phenomenon was more evident with L-histidine ligand due to more hydrophilic character, leading to an improved selectivity of L-histidine-modified chromatographic monolith, allowing the product recovery with 99% of purity (RNA removal). With the 1-benzyl- L-histidine ligand, stronger and less selective interactions with the nucleic acids were observed due to the additional hydrophobicity associated with the phenyl aromatic ring. Optimization of sample displacement chromatography parameters (especially (NH4 )2 SO4 concentration) at slightly acidic pH enabled excellent isolation of pDNA, by the removal of RNA in a negative mode, with binding capacities above 1.5 mg pDNA per mL of chromatographic support.

2017Journal article

Restricted access

Brain-Targeted Delivery of Pre-miR-29b Using Lactoferrin-Stearic Acid-Modified-Chitosan/Polyethyleneimine Polyplexes

Publication . Pereira, Patrícia; Barreira, Maria; Cruz, Carla; Tomás, Joana; Luís, Ângelo; Pedro, Augusto; Queiroz, João; Sousa, Fani

The efficacy of brain therapeutics is largely hampered by the presence of the blood-brain barrier (BBB), mainly due to the failure of most (bio) pharmaceuticals to cross it. Accordingly, this study aims to develop nanocarriers for targeted delivery of recombinant precursor microRNA (pre-miR-29b), foreseeing a decrease in the expression of the BACE1 protein, with potential implications in Alzheimer's disease (AD) treatment. Stearic acid (SA) and lactoferrin (Lf) were successfully exploited as brain-targeting ligands to modify cationic polymers (chitosan (CS) or polyethyleneimine (PEI)), and its BBB penetration behavior was evaluated. The intracellular uptake of the dual-targeting drug delivery systems by neuronal cell models, as well as the gene silencing efficiency of recombinant pre-miR-29b, was analyzed in vitro. Labeled pre-miR-29b-CS/PEI-SA-Lf systems showed very strong fluorescence in the cytoplasm and nucleus of RBE4 cells, being verified the delivery of pre-miR-29b to neuronal cells after 1 h transfection. The experiment of transport across the BBB showed that CS-SA-Lf delivered 65% of recombinant pre-miR-29b in a period of 4 h, a significantly higher transport ratio than the 42% found for PEI-SA-Lf in the same time frame. Overall, a novel procedure for the dual targeting of DDS is disclosed, opening new perspectives in nanomedicines delivery, whereby a novel drug delivery system harvests the merits and properties of the different immobilized ligands.

2020Journal article

Open access